JP4587652B2 - Novel flavonoid compounds and their use - Google Patents

Description

  The present invention relates to a novel flavonoid compound and use thereof. In particular, the present invention relates to a flavonoid compound that easily exhibits activity in cells and use thereof.

  Flavonoids are contained in a wide range of vegetable foods such as vegetables and fruits and have various physiological activities.

  As an example of physiological activity, flavonoids have an antioxidant action to eliminate active oxygen. For this reason, flavonoids can suppress damage due to, for example, DNA oxidation, and as a result, have an action of preventing carcinogenesis (for example, Non-Patent Document 1). Further, where inflammation occurs due to an excess of active oxygen, flavonoids have an anti-inflammatory activity because they have an antioxidant action (for example, Non-Patent Document 2). Furthermore, flavonoids can prevent arteriosclerosis caused by oxidized LDL by inhibiting peroxidation of low density lipoprotein (LDL) in blood (for example, Non-patent Document 3).

  Heterocyclic amines are carcinogens generated by heating proteinaceous foods, and are metabolized and activated by 1A of the liver metabolic enzyme cytochrome P450. Flavonoids inhibit the activity of the enzyme. It is also known to prevent carcinogenesis (for example, Non-Patent Document 4).

  In addition, some flavonoids have been reported to exhibit an estrogen-like action and prevent breast cancer (for example, Non-Patent Document 5).

  In addition, flavonoids suppress dioxin toxicity at an early stage of toxic expression that activates aryl hydrocarbon receptors (for example, Patent Document 1).

  Furthermore, flavonoids have also been reported to stop the growth by regulating the cell cycle of cancer cells (for example, Non-Patent Document 6).

  In epidemiological studies, a correlation has been found between the intake of flavonoids and mortality due to coronary heart disease or cancer (for example, Non-Patent Document 7).

Flavonoids exist mainly as glycosides in foods, but most of them are hydrolyzed in the small intestine and taken up into small intestinal cells as aglycone. Many of the flavonoids taken up into small intestinal cells are conjugated to glucuronic acid or sulfuric acid and released into the small intestinal cavity or blood. In particular, since flavonoids having a hydroxyl group are susceptible to conjugation, they are rapidly excreted from the cells and further excreted in urine or bile. Therefore, in order to maintain a flavonoid concentration at which sufficient physiological activity can be expressed in cells, it is necessary to ingest a large amount of flavonoids.
JP 2001-348382 A (paragraph 0011) Free Radic.Res.36,2002,307-316 Food Factors for Cancer Prevention (Springer), p468-471,1997 Biochem. Pharmacol. 39, 1990, 1743-50; J. Clin. Invest. 88, 1991, 1785-92 Mutat.Res.379,1997,21-32 Food Chem. Toxic. 38,2000,555-64 Food Factors for Cancer Prevention (Springer), p623-626,1997 Lancet. 342, 1993, 1007-11; J. Natl. Cancer Inst. 92, 2000, 154-60

  The present invention provides a novel flavonoid compound that maintains the physiological activity possessed by flavonoids and that easily expresses the physiological activity in cells, a method for producing the compound, and a pharmaceutical or food composition containing the compound. The main purpose.

  In order to achieve the above-mentioned object, the present inventor obtained the following knowledge as a result of repeated research.

  That is, the general formula (1)

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a hydroxyl group or a lower alkoxy group. R ¹⁰ And R ¹¹ together represent a hydrogen atom or an oxo group together, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a single bond or a double bond (positions 1 to 4 of the chroman skeleton). The position of the place is indicated by a number in the formula))
The compound represented by
The following general formula (2)

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a hydroxyl group or a lower alkoxy group. R ¹⁰ And R ¹¹ together represent a hydrogen atom or an oxo group together, and the bond between the 2-position and 3-position of the chroman skeleton represents a single bond or a double bond.)
And a physiological activity equivalent to that of the compound represented by (corresponding flavonol compound having a hydroxyl group instead of the amino group at the 3-position of the chroman skeleton of the compound of the general formula (1)) and being easily taken up into cells, It is difficult to be conjugated with glucuronic acid or the like.

  Normally, the “chroman skeleton” does not include those in which the bond between the 2-position and the 3-position is a double bond, but the “chroman skeleton” in the present invention includes such a thing.

  This invention is completed based on the said knowledge, The following compound, its manufacturing method, and the pharmaceutical or foodstuff composition which uses this compound as an active ingredient are provided.

  Item 1. The compound represented by the following general formula (1).

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a hydroxyl group or a lower alkoxy group. R ¹⁰ And R ¹¹ together represent a hydrogen atom or an oxo group together, and the bond between the 2-position and 3-position of the chroman skeleton represents a single bond or a double bond.)
Item 2. In the general formula (1), R ² , R ⁴ and R ⁷ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together The compound according to Item 1, wherein the compound represents an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

Item 3. In the general formula (1), R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together represent oxo. Item 2. The compound according to item 1, wherein the compound represents a group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

Item 4. In the general formula (1), R ² and R ⁴ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together The compound according to Item 1, wherein the compound represents an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

Item 5. In the general formula (1), R ² , R ⁷ and R ⁸ represent a hydroxyl group, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together The compound according to Item 1, wherein the compound represents an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

Item 6. In the general formula (1), R ² , R ⁴ , R ⁷ and R ⁹ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁸ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together. The compound according to Item 1, wherein the compound represents an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

Item 7. In the general formula (1), R ² , R ⁴ , R ⁷ and R ⁸ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together. The compound according to Item 1, wherein the compound represents an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

Item 8. In the general formula (1), R ² , R ⁴ , R ⁶ , R ⁷ and R ⁸ represent a hydroxyl group, R ¹ , R ³ , R ⁵ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together. The compound according to Item 1, wherein the compound represents an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

Item 9. In the general formula (1), R ² , R ⁴ and R ⁸ represent a hydroxyl group, R ⁷ represents a methoxy group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, R ¹⁰ And R ¹¹ together represent an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

Item 10. In the general formula (1), R ² , R ⁴ and R ⁷ represent a hydroxyl group, R ⁸ represents a methoxy group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, R ¹⁰ And R ¹¹ together represent an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

  Item 11. The following general formula (1)

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a hydroxyl group or a lower alkoxy group. R ¹⁰ And R ¹¹ together represent a hydrogen atom or an oxo group together, and the bond between the 2-position and 3-position of the chroman skeleton represents a single bond or a double bond.)
A compound represented by the following general formula (2):

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a hydroxyl group or a lower alkoxy group. R ¹⁰ And R ¹¹ together represent a hydrogen atom or an oxo group together, and the bond between the 2-position and 3-position of the chroman skeleton represents a single bond or a double bond.)
A step of incubating the compound of the present invention with a mammalian small intestinal surface cell homogenate; and a step of recovering the compound of the general formula (1).

Item 12. In the general formula (1), R ² , R ⁴ and R ⁷ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ represent A method for producing a compound which together represents an oxo group, and wherein the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond, wherein in the general formula (2), R ² , R ⁴ and R ⁷ represents a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the 2nd position of the chroman skeleton And a step of incubating a compound in which the bond between position 1 and position 3 represents a double bond with a mammalian small intestine surface cell homogenate; in the above general formula (1), R ² , R ⁴ and R ⁷ represent a hydroxyl group; , R ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and between the 2nd and 3rd positions of the chroman skeleton Double bond Manufacturing method comprising the step of recovering the compound representing a slip.

  Item 13. The following general formula (1)

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a hydroxyl group or a lower alkoxy group. R ¹⁰ And R ¹¹ together represent a hydrogen atom or an oxo group together, and the bond between the 2-position and 3-position of the chroman skeleton represents a single bond or a double bond.)
A compound represented by the following general formula (2):

(Wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are the same or different and each represents a hydrogen atom, a hydroxyl group or a lower alkoxy group. R ¹⁰ And R ¹¹ together represent a hydrogen atom or an oxo group together, and the bond between the 2-position and 3-position of the chroman skeleton represents a single bond or a double bond.)
And a step of recovering the compound of the above general formula (1).

  Item 14. Item 14. The method according to Item 13, wherein the step of leaving the compound of the general formula (2) in the presence of ammonia is performed under pressure.

Item 15. In the compound represented by the general formula (1), R ² , R ⁴ and R ⁷ in the general formula (1) represent a hydroxyl group, and R ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ Is a compound in which R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond, In the above general formula (2), R ² , R ⁴ and R ⁷ represent a hydroxyl group, and R ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ represent a hydrogen atom. Item 15. The method according to Item 13 or 14, wherein R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

  Item 16. Item 11. A gene oxidation inhibitor comprising the compound according to any one of Items 1 to 10.

  Item 17. Item 11. A dioxin toxicity expression inhibitor comprising the compound according to any one of Items 1 to 10.

  Item 18. Item 11. An anticancer agent comprising the compound according to any one of Items 1 to 10.

  Item 19. Item 11. An immunodeficiency preventive agent comprising the compound according to any one of Items 1 to 10.

  Item 20. Item 11. A teratogenicity preventive agent comprising the compound according to any one of Items 1 to 10.

  Item 21. Item 11. An anti-inflammatory agent comprising the compound according to any one of Items 1 to 10.

  Item 22. Item 11. A prophylactic or therapeutic agent for arteriosclerosis comprising the compound according to any one of Items 1 to 10.

  Item 23. Item 11. A preventive or therapeutic agent for diabetes comprising the compound according to any one of Items 1 to 10.

  Item 24. Item 11. A composition for suppressing gene oxidation, comprising the compound according to any one of Items 1 to 10 as an active ingredient.

  Item 25. Item 11. A composition for suppressing the expression of dioxin toxicity, comprising the compound according to any one of Items 1 to 10 as an active ingredient.

  Item 26. Item 11. An anticancer composition comprising the compound according to any one of Items 1 to 10 as an active ingredient.

  Item 27. Item 11. A composition for preventing immunodeficiency, comprising the compound according to any one of Items 1 to 10 as an active ingredient.

  Item 28. Item 11. A composition for preventing teratogenicity comprising the compound according to any one of Items 1 to 10 as an active ingredient.

  Item 29. Item 11. An anti-inflammatory composition comprising the compound according to any one of Items 1 to 10 as an active ingredient.

  Item 30. Item 11. A composition for preventing or treating arteriosclerosis, comprising the compound according to any one of Items 1 to 10 as an active ingredient.

  Item 31. Item 11. A composition for preventing or treating diabetes comprising the compound according to any one of Items 1 to 10 as an active ingredient.

  Item 32. Item 11. A cancer prevention method comprising administering the compound according to any one of Items 1 to 10 to a human.

  Item 33. Item 11. A method for preventing immunodeficiency, comprising administering the compound according to any one of Items 1 to 10 to a human.

  Item 34. Item 11. A method for preventing teratogenicity, comprising administering the compound according to any one of Items 1 to 10 to a human.

  Item 35. Item 11. A method for preventing or treating inflammation, wherein the compound according to any one of Items 1 to 10 is administered to a human.

  Item 36. Item 11. A method for preventing or treating arteriosclerosis, comprising administering the compound according to any one of Items 1 to 10 to a human.

  Item 37. Item 11. A method for preventing diabetes, comprising administering the compound according to any one of Items 1 to 10 to a human.

  According to the present invention, there is provided a novel flavonoid compound that maintains the physiological activity possessed by flavonoids and easily expresses the physiological activity in cells.

  Furthermore, the compound of the present invention is represented by the above general formula (1), and has the same physiological activity as the corresponding flavonol compound represented by the above general formula (2). In addition, since the compound of the present invention has an amino group, it is easily taken up into cells and hardly conjugated with glucuronic acid or the like, so that it can be present in cells at a high concentration. In this respect, it is more useful as an active ingredient for pharmaceuticals, health foods and the like than the corresponding flavonol compound represented by the general formula (2).

Hereinafter, the present invention will be described in detail.
(I) Compound of the Present Invention The compound of the present invention is a novel compound not described in any literature, and is represented by the general formula (1)
It is a compound represented by these.

  Examples of the lower alkoxy group include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, sec-butoxy group, tert-butoxy group, n-pentyl group, isopentyl group, neopentyl group, tert -A linear or branched alkoxy group having about 1 to 5 carbon atoms, particularly about 1 to 4 carbon atoms, such as a pentyl group.

Among the compounds of the general formula (1), the following compounds are particularly preferable.
R ² , R ⁴ and R ⁷ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together form an oxo group. A compound in which the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

This compound is a kaempferol known as one of flavonols (in the above general formula (2), R ² , R ⁴ and R ⁷ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond) In which the hydroxyl group at the 3-position of the chroman skeleton is an amino group.
R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and a chroman skeleton A compound in which the bond between the 2-position and the 3-position represents a double bond.

This compound is a flavonol known as one of flavonols (in the above general formula (2), R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ And R ⁹ represents a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2- and 3-positions of the chroman skeleton represents a double bond). The coordinate hydroxyl group is an amino group.
R ² and R ⁴ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together form an oxo group. A compound in which the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

This compound is galangin known as one of flavonols (in the above general formula (2), R ² and R ⁴ represent a hydroxyl group, and R ¹ , R ³ , R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond) The 3-position hydroxyl group of the chroman skeleton is an amino group.
R ² , R ⁷ and R ⁸ represent a hydroxyl group, R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together form an oxo group. A compound in which the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

This compound is a fisetin known as one of flavonols (in the general formula (2), R ² , R ⁷ and R ⁸ represent a hydroxyl group, and R ¹ , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond) The 3-position hydroxyl group of the chroman skeleton is an amino group.
R ² , R ⁴ , R ⁷ and R ⁹ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁸ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together form an oxo group. A compound in which the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

This compound is Morin known as one of flavonols (in the above general formula (2), R ² , R ⁴ , R ⁷ , R ⁹ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁸ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond) The 3-position hydroxyl group of the chroman skeleton is an amino group.
R ² , R ⁴ , R ⁷ and R ⁸ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together form an oxo group. A compound in which the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.

This compound is quercetin known as one of flavonols (in the above general formula (2), R ² , R ⁴ , R ⁶ , R ⁷ and R ⁸ represent a hydroxyl group, R ¹ , R ³ , R ⁵ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond) The 3-position hydroxyl group of the chroman skeleton is an amino group.
R ² , R ⁴ , R ⁶ , R ⁷ and R ⁸ represent a hydroxyl group, R ¹ , R ³ , R ⁵ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together form an oxo group. A compound in which the bond between position 2 and position 3 of the chroman skeleton represents a double bond.

This compound is myricetin known as one of flavonols (in the above general formula (2), R ² , R ⁴ , R ⁶ , R ⁷ , R ⁸ represent a hydroxyl group, R ¹ , R ³ , R ⁵ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond) The 3-position hydroxyl group of the chroman skeleton is an amino group.
R ² , R ⁴ and R ⁸ represent a hydroxyl group, R ⁷ represents a methoxy group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together A compound in which the oxo group is represented and the bond between the 2nd and 3rd positions of the chroman skeleton is a double bond.

This compound includes tamarixetin known as one of flavonols (in the above general formula (2), R ² , R ⁴ and R ⁸ represent a hydroxyl group, R ⁷ represents a methoxy group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton is a double bond In which the hydroxyl group at the 3-position of the chroman skeleton is an amino group.
R ² , R ⁴ and R ⁷ represent a hydroxyl group, R ⁸ represents a methoxy group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, and R ¹⁰ and R ¹¹ together A compound in which the oxo group is represented and the bond between the 2nd and 3rd positions of the chroman skeleton is a double bond.

This compound is an isorhamnetin known as one of flavonols (in the above general formula (2), R ² , R ⁴ and R ⁷ represent a hydroxyl group, R ⁸ represents a methoxy group, R ¹ , R ³ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton is a double bond In which the hydroxyl group at the 3-position of the chroman skeleton is an amino group.

In particular, in the general formula (1), R ² , R ⁴ and R ⁷ represent a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ Are preferred, which together represent an oxo group, and the bond between the 2-position and the 3-position of the chroman skeleton represents a double bond.
Action and Use of the Compound of the Present Invention The compound of the present invention has a physiological activity equivalent to that of flavonoids.

  Specifically, the compound of the present invention has an activity of suppressing DNA oxidation by AAPH, which is a free radical generating compound. Non-patent document 1 describes that oxidation of DNA induces carcinogenesis. Further, the compound of the present invention has an activity of suppressing DNA mutation caused by heterocyclic amines (for example, Trp-P-2) which are food-derived DNA mutagens. From these facts, the compound of the present invention can prevent carcinogenesis and can be suitably used as an anticancer agent, particularly a cancer preventive agent.

  Also, when deoxyguanosine in DNA is oxidized to 8-oxodeoxyguanosine, it is read as adenosine or the like during gene transcription. It has been reported to induce mutations and cause arteriosclerosis and diabetes (Mutat. Res., 267, 1992, 277-290; Free Radic. Biol. Med., 26, 1999, 202-226. ). Since the compound of the present invention suppresses such oxidation by AAPH, it can be suitably used as a prophylactic agent for arteriosclerosis and diabetes.

  Dioxin is activated by binding to aryl hydrogen hydrocarbon receptor (AhR) to express various genes, and as a result, induces carcinogenesis, immunodeficiency, teratogenicity, etc. (Patent Document 1). The compound of the present invention suppresses the toxicity of dioxin by suppressing the activation of AhR. Thus, the compound of the present invention can be suitably used as an agent for preventing, treating or ameliorating diseases caused by dioxins, such as an anticancer agent (particularly a cancer preventing agent), an immunodeficiency preventing agent or a teratogenicity preventing agent.

  The physiological activity of the compound of the present invention described above is slightly inferior to that of the corresponding flavonol compound, but is substantially equivalent.

  In addition, where inflammation occurs due to an excess of active oxygen, the compound of the present invention has an antioxidant action, and thus is considered to have an anti-inflammatory action. From this, it is considered that the compound of the present invention can be suitably used as an anti-inflammatory agent.

  Further, since the compound of the present invention has an antioxidant action, it is considered to have an action of suppressing arteriosclerosis caused by oxidized LDL by suppressing peroxidation of low density lipoprotein (LDL) in blood. It is done. From this fact, it is considered that the compound of the present invention can be used as an arteriosclerosis preventive agent or an arteriosclerosis treatment or amelioration agent.

  In addition, the compound of the present invention exhibits an estrogen-like action and is considered to suppress breast cancer. From this, it is considered that the compound of the present invention can be used as an anticancer agent, particularly as a preventive, therapeutic or ameliorating agent for breast cancer.

  Moreover, it is thought that the compound of this invention has a blood glucose level lowering effect. From this, it is considered that the compound of the present invention can be used as a preventive, therapeutic or ameliorating agent for diabetes.

  In addition, the compound of the present invention is considered to have an action of regulating the cell cycle of cancer cells to stop their proliferation. From this, it is considered that the compound of the present invention can be used as an anticancer agent.

  Conventionally known flavonol compounds are difficult to enter into cells and are susceptible to conjugation with glucuronic acid and the like, so that it is necessary to take them in large quantities in order to express their activity in the cells, and it is difficult to use them as pharmaceuticals.

  On the other hand, since the compound of the present invention has an amino group, it is easily taken into small intestine cells, and easily migrates from the small intestinal cells to the serosa side (blood vessel side). Furthermore, since the compound of the present invention is less susceptible to conjugation with sulfuric acid or glucuronic acid than the corresponding flavonol, it is difficult to excrete into urine or bile, and as a result, it is difficult to excrete from cells.

From these facts, the compound of the present invention can be present in small intestinal cells or cells of other tissues at a concentration sufficient to express physiological activity by administration in a small amount, and thus is easy to use as an active ingredient of pharmaceuticals. . Further, since the compound of the present invention expresses physiological activity in a relatively small amount, the health food containing the compound of the present invention as an active ingredient becomes relatively inexpensive.
(II) Pharmaceutical Composition / Food Composition The composition of the present invention is a composition comprising the compound represented by the general formula (1) as an active ingredient, which inhibits gene oxidation, dioxin toxicity, and activity. It can be used to remove oxygen and suppress peroxidation of blood LDL.

  Specifically, it can be used for preventing carcinogenesis caused by gene oxidation or carcinogenesis induced by dioxin. It can also be used to prevent arteriosclerosis due to gene oxidation. It can also be used to prevent or treat (including improvement) diabetes caused by gene oxidation. It can also be used to prevent immune deficiency and teratogenicity induced by dioxins. Moreover, it can be used for prevention or treatment (including improvement) of inflammation by eliminating active oxygen. It can also be used for the prevention or treatment (including improvement) of arteriosclerosis by inhibiting the peroxidation of blood LDL.

  The composition of the present invention may be a pharmaceutical composition or a food composition. The pharmaceutical composition may consist only of the compound of the present invention, and is a mixture of pharmaceutically acceptable carriers (activators, extenders, binders, lubricants, etc.) and conventional additives. It may be. This pharmaceutical composition is prepared in a form (oral administration such as tablets, pills, capsules, powders, granules, syrups, etc .; parenteral administration such as injections, drops, external preparations, suppositories), etc. Depending on the dosage, it can be administered orally or parenterally. In addition, the dosage varies depending on the type of active ingredient, age of the subject to be administered or patient, body weight, symptoms, and cannot be defined unconditionally, but as a daily dosage, in the case of oral administration, about 0.1 to 5000 mg (particularly about 0.1 to 500 mg), In the case of parenteral administration, about 0.01 to 500 mg (particularly about 0.01 to 50 mg) can be administered once or several times a day.

  The kind of food contained in the food composition is not particularly limited. For example, it can be made into the composition which added the compound of this invention etc. to foods, such as sweets, such as cookies, jelly; drinks, such as juice and tea;

The content of the compound of the present invention in the food composition may be included so that the daily intake is about 0.01 to 100 mg.
(III) Prevention method / treatment method The method of the present invention comprises a cancer prevention method, a similar immunodeficiency prevention method, and a similar teratogenicity prevention method in which the compound represented by the general formula (1) of the present invention is administered to humans. And a similar method for preventing or treating inflammation, a similar method for preventing or treating arteriosclerosis, and a method for preventing diabetes.

  Humans to be administered are not particularly limited and are healthy people; employees of garbage incineration facilities that are susceptible to diseases caused by dioxins; residents near garbage incineration facilities; smokers who are susceptible to cancer, cirrhosis patients, For patients with colorectal polyps; patients with hypertension.

The administration method is not particularly limited. In addition, the daily dose may be about 0.1 to 5000 mg (particularly about 0.1 to 500 mg) for oral administration and about 0.01 to 500 mg (particularly about 0.01 to 50 mg) for parenteral administration.
(IV) Method for producing the compound of the present invention
First Production Method The compound of the present invention is a compound represented by the above general formula (2) (a corresponding compound in which the 3-position amino group of the chroman skeleton of the compound represented by the above general formula (1) is substituted with a hydroxyl group. ) Is incubated with, for example, a mammalian small intestine surface cell homogenate, and the produced compound represented by the above general formula (1) is recovered.
<Raw compound>
In the present invention, the compound of the general formula (2) is used as a raw material. These compounds are commercially available from Wako Pure Chemicals, Nacalai Tesque, Sigma, Funakoshi, Kurihara Sangyo, Extrasintés (France), and the like.
<Small intestinal surface cell homogenate>
The type of mammal is not particularly limited, and it is sufficient to use easily available animals such as rats, mice, rabbits, pigs, dogs, etc. However, it is preferable to use rats from the viewpoint of easily obtaining high specific activity. Of the small intestine surface cells, surface cells near the duodenum and the duodenum of the jejunum are more preferable in that a high specific activity can be obtained.

The small intestine surface cell homogenate can be obtained, for example, by the following method. First, after reversing the small intestine extracted from an animal, intestinal surface cells are scraped off with a glass plate or the like. The obtained surface cells are suspended in a buffer having a pH of about 4 to 7.5 such as a phosphate buffer. This cell suspension is homogenized with a homogenizer and diluted with the same buffer so that the protein amount is about 0.1 to 10 mg / ml, particularly about 2 to 4 mg / ml, whereby a small intestinal surface cell homogenate is obtained.
<Metabolism of raw material compounds>
A solution prepared by dissolving a raw material compound in an appropriate solvent (water containing alcohol, methanol, ethanol, propyl alcohol, isopropyl alcohol, ethyl acetate, or the like) is added to a small intestine surface cell homogenate at a final concentration of about 1 to 100 μM, particularly 20 to Add to about 30 μM and stir.

  This is incubated at about 25 to 50 ° C., particularly about 35 to 38 ° C. for about 5 to 480 minutes, particularly about 60 to 120 minutes. Thereby, a compound in which the 3-position hydroxyl group of the chroman skeleton is converted to an amino group in the general formula (1) is generated.

Subsequently, the target compound may be recovered and purified according to a conventional method. For example, the reaction solution after incubation can be centrifuged and the supernatant collected, and then purified by a method such as HPLC.
Second Production Method The compound represented by the above general formula (1) of the present invention is produced by leaving the compound represented by the above general formula (2) in the presence of ammonia and then generating the above general formula (1). It can obtain also by collect | recovering the compound represented by these.

  Ammonia may be used as aqueous ammonia or as a lower alcohol solution of ammonia. Examples of the lower alcohol include methanol, ethanol, n-butyl alcohol, iso-butyl alcohol, n-propyl alcohol, iso-propyl alcohol, tert-propyl alcohol and the like. In particular, ethanol is preferable. The concentration of ammonia in the reaction solution is usually about 10 to 18N, and particularly preferably about 18N which is a saturated concentration.

  Further, the compound of the above general formula (2) may be usually dissolved in an ammonia solution at a concentration of about 10 mg / l to 10 g / l, particularly about 1 to 10 g / l. The standing is preferably performed at a temperature of usually about 5 to 80 ° C., particularly about 25 to 80 ° C., usually for about 1 to 24 hours, particularly about 5 to 24 hours.

  It is preferable to leave the raw material compound in the presence of ammonia as described above under pressure. For example, the pressure is preferably about 5 to 10 atm, and more preferably about 7 to 10 atm. Pressurization within this range can greatly improve the yield of the target compound.

  Moreover, it is preferable to add ammonium chloride normally about 1-100 g / l, especially about 30-100 g / l irrespective of the presence or absence of pressurization. Thereby, the yield of the target compound can be improved.

The flavonoid compound of the present invention thus produced may be recovered by HPLC or the like.
EXAMPLES Hereinafter, although an Example and a test example are shown and this invention is demonstrated in detail, this invention is not limited to these Examples.

Production of 3-position amino form of kaempferol
Preparation of rat small intestinal surface cell homogenate
After removing the small intestine from Wister / ST rats (6 weeks old) (JAPAN ALC. Inc.) and refluxing with cold PBS (phosphate-buffered-saline, pH 7.4), the contents were removed. Cut open, washed in cold PBS and inverted. After removing moisture with filter paper, the small intestine surface cells were taken out using a glass plate. Cold PBS was added thereto, and the mixture was homogenized with a Potter-Elvehjem type homogenizer in ice. Furthermore, the protein amount of this homogenate was measured by the method of Lowry et al. Using bovine serum albumin as a standard substance, and diluted with cold PBS to obtain 4 mg protein / ml, thereby obtaining rat small intestinal surface cell homogenate. It was.
Production of the Compound of the Present Invention The rat small intestine surface cell homogenate was preincubated at 37 ° C. for 1 minute, and then kaempferol (in the above general formula (2), R ² , R ⁴ and R ⁷ represent a hydroxyl group, R A DMSO solution of a compound in which ¹ , R ³ , R ⁵ , R ⁶ , R ⁸ and R ⁹ represent hydrogen atoms (manufactured by Wako Pure Chemical Industries, Ltd.) was added to a final concentration of 30 μM.

  After incubation at 37 ° C. for 60 minutes, 2.0 ml of ethyl acetate was added and the reaction was stopped by cooling with ice.

  The reaction solution was thoroughly stirred with a stirrer (vortex mixer) and then centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected in an eggplant type flask. This extraction with ethyl acetate was repeated three times, and then the ethyl acetate was removed with an evaporator. This was redissolved in methanol and recovered, and again dried under nitrogen gas. The residue was redissolved in the HPLC mobile phase, filtered with a membrane filter having a pore size of 0.2 μm, and then subjected to preparative HPLC to fractionate the metabolite-containing fraction. The collected fraction was dried.

  The HPLC conditions are as follows.

Pump: SHIMAZU LC-5A
Detector: SHIMAZU SPD-2A
Detection: UV250nm
Column: SHISEIDO CAPCELL PAK C18
UG80 φ10 × 250nm
Mobile phase: 0.1% TFA: Methanol = 40:60
Flow rate: 2.0ml / min Injection volume: 200μl
Identification of product compounds
<TLC>
The metabolite and kaempferol were developed in a developing solution having a composition of 1% TFA solution: methanol = 4: 6 using Whatman KC18F reverse phase plate. The Rf value of the metabolite was 0.43, and the Rf value of kaempferol was 0.35. It turns out that both are different compounds.
<HPLC-DAD>
The UV spectrum of the metabolite and kaempferol was measured under the following conditions.
Pump: HITACHI L-7100
Detector: HITACHI L-7455
Detection: UV250-450nm
Column: SHISEIDO CAPCELL PAK C18, UG120φ4.6 × 250mm
Mobile phase: 0.1% TFA: Methanol = 45:55
Flow rate: 1.0 ml / min Injection volume: 10 μl
The UV spectrum is shown in FIG. The metabolite λmax was found at 260 nm and 365 nm, and kaempferol λmax was found at 262 nm and 360 nm. Thus, there was a slight shift in the peak due to metabolism. Moreover, the peak height on the long wavelength side was slightly smaller for metabolites than kaempferol.

From these results, it was considered that the metabolite was a kaempferol derivative that retained the kaempferol skeleton.
<FT-IR>
IR spectra of metabolites and kaempferol were measured by FT-IR (SHIMAZU SSU-8000). The measurement was performed by a diffuse reflection method with a resolution of 4 cm ⁻¹ and an accumulation number of 400 times. The sample was mixed with KBr powder and used for measurement.

The IR spectrum is shown in FIG. IR spectra of the metabolites absorption characteristic functional groups in the wave number region of 3800 cm ^-1 from 1500 cm ^-1 is not found, also it was similar to the IR spectrum of kaempferol in fingerprint region of 1500 cm ^-1 from 750 nm ^-1 . This also shows that the metabolite is a compound very similar to kaempferol in terms of chemical structure.
<NMR>
With respect to the metabolite and kaempferol, one-dimensional NMR ( ¹ H-NMR, ¹³ C-NMR, DEPT) spectra were measured with an FT-NMR system. Metabolites and kaempferol were dissolved in methanol-d ₄ (CD ₃ OD) for analysis.

^{The 1} H-NMR spectrum is shown in FIG. 3A is a spectrum of kaempferol, and FIG. 3B is a spectrum of a metabolite. As apparent from FIG. 3, in kaempferol, doublet signals were observed at positions of 6.1 ppm, 6.3 ppm, 6.9 ppm, and 8.0 ppm. Even for metabolites, doublet signals were observed at 6.1, 6.3, 6.9, and 7.7 ppm.

^{The 13} C-NMR spectrum is shown in FIG. 4A shows the spectrum of kaempferol, and FIG. 4B shows the spectrum of the metabolite. As is clear from FIG. 4, in Kaempferol, 13 signals were observed in positions near 94 ppm, 99 ppm, 104 ppm, 116 ppm, 123 ppm, 130 ppm, 137 ppm, 148 ppm, 158 ppm, 160 ppm, 162 ppm, 165 ppm, and 177 ppm. . In contrast, in the metabolite, nine signals were observed at positions of 94 ppm, 99 ppm, 116 ppm, 124 ppm, 130 ppm, 158 ppm, 160 ppm, 162 ppm, and 165 ppm.

Proton signal shifts due to the metabolism of kaempferol and changes in the carbon signals at positions 2, 3, 4 and 10 suggest that some changes have occurred at position 3.
<Mass spectrometry>
1) Low-resolution FAB-MS measurements were performed on kaempferol and metabolites under the following conditions.

Ionization conditions: Ionization method FAB
Ion polarity Positive
Matrix Glycerol Measurement conditions: Measurement range m / z 10-1000 (magnetic field scan)
Acceleration voltage 10kV
Scan speed 12sec / decade
The spectrum of kaempferol is shown in FIG. 5, and the spectrum of the metabolite is shown in FIG. As shown in FIG. 5, in kaempferol, m / z 287 [M + H] ⁺ judged as a molecular ion peak was detected. As shown in FIG. 6, a plurality of other component peaks were detected in the metabolite, but a peak (m / z 286) supporting the estimated molecular weight 285 was detected with an intensity of about 10%.
2) The molecular ion peak of the metabolite confirmed in the low resolution MS measurement was subjected to the high resolution FAB-MS measurement under the following conditions.

Ionization conditions: Ionization method FAB
Ion polarity Positive
Matrix Glycerol Measurement conditions: Measurement range m / z 278-332 (magnetic field scan)
Standard substance PEG400
Scan speed 100sec / decade
The precise mass results and composition calculation results of the metabolites are shown in Table 1 below.

From Table 1, for the metabolite, the composition formula C ₁₅ H ₁₂ O ₅ N ( ¹ H addition) when the hydroxyl group of kaempferol was substituted with an amino group was obtained with an error of −1.1 ppm / −0.3 mmu.

  As a result of the above NMR and mass spectrometry, the metabolite of kaempferol is a compound (compound represented by the following general formula (3)) in which the hydroxyl group at the 3-position of the chroman skeleton of kaempferol is substituted with an amino group. It was concluded.

In Production Example 1 of other flavonoid compounds , instead of kaempferol as a raw material compound, flavonol ( Preparation Example 2 ), galanthin ( Preparation Example 3 ), fisetin ( Preparation Example 4 ), Morin ( Preparation Example 5 ), A metabolite was obtained in the same manner as in Example 1 except that quercetin ( Preparation Example 6 ), myricetin ( Preparation Example 7 ), tamarixetine ( Preparation Example 8 ), and isorhamnetin ( Preparation Example 9 ) were used.

Moreover, in Example 1, it replaces with kaempferol as a raw material compound, and it is the same as that of Example 1 except having used dacetetine ( Preparation Example 10 ), robinetin ( Preparation Example 11 ), and quercetagetin ( Preparation Example 12 ), respectively. Metabolites were obtained. In the above general formula (2), daticetine is represented by R ² , R ⁴ and R ⁹ representing a hydroxyl group, R ¹ , R ³ , R ⁵ , R ⁶ , R ⁷ and R ⁸ representing a hydrogen atom, R ¹⁰ and R ¹¹ is a compound in which ¹¹ represents an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond. In the above general formula (2), robinetine is represented by R ² , R ⁶ , R ⁷ and R ⁸ representing a hydroxyl group, R ¹ , R ³ , R ⁴ , R ⁵ and R ⁹ representing a hydrogen atom, R ¹⁰ and R ¹¹ is a compound in which ¹¹ represents an oxo group and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond. Quercetagetin in the above general formula (2), R ² , R ³ , R ⁴ , R ⁷ and R ⁸ represent a hydroxyl group, R ¹ , R ⁵ , R ⁶ and R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ is a compound in which ¹¹ represents an oxo group and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond.

  The metabolite is a compound in which the hydroxyl group of the compound of the general formula (1) is changed to an amino group because it is eluted at a position different from the starting compound in LC / MS LC and is 1 mass unit smaller in MS. It was judged. The general formula of the obtained compound is shown in Table 2 below.

Production of compounds of the present invention by organic synthesis
Kaempferol (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 18N ammonia water so as to have a concentration of 500 mg / l, and left at room temperature for 20 hours under 10 atm. The target compound was extracted from the resulting solution with ethyl acetate, and the extract was separated and purified by HPLC in the same manner as in Example 1. The product was identified by the same method as in Preparation Examples 2 to 8, and in the general formula (1), R ² , R ⁴ and R ⁷ represent a hydroxyl group, and R ¹ , R ³ and R ⁵ , R ⁶ , R ⁸ , R ⁹ represent a hydrogen atom, R ¹⁰ and R ¹¹ together represent an oxo group, and the bond between the 2nd and 3rd positions of the chroman skeleton represents a double bond It was determined that

  The yield was 10%.

In the production the third embodiment of the present invention compounds according to an organic synthesis, except that the reaction was carried out with ammonia at atmospheric pressure was carried out in the same manner as in Example 3. The product yield was 3%.

In vitro antioxidant activity
<DPPH method>
Antioxidant activity by kaempferol and metabolites was evaluated by measuring their ability to reduce DPPH (diphenylpicrylhydrazyl). To a mixed solution of 500 μl of acetate buffer (pH 5.5), 500 μl of ethanol, and 250 μl of 0.5 mM DPPH solution, 10 μl of Kaempferol methanol solution and metabolite methanol solution were added, respectively. After 5 minutes, the absorbance at 517 nm, which is the maximum absorption wavelength of DPPH radical, was measured. The DPPH radical is a stable radical and is pink in the radical state, but when this radical is captured, it becomes a colorless leuco type (Nature, 181, 1958, 1199-1200). Therefore, the lower the absorbance at 517 nm, the more the DPPH radical is reduced, that is, the stronger the antioxidant activity by kaempferol or metabolites.

  The concentration of kaempferol and metabolites in the methanol solution was changed to 1, 2, 3, 4 and 5 mM. FIG. 7 shows a graph in which DPPH concentration (%) is plotted against kaempferol or metabolite concentration. FIG. 7A shows the result of kaempferol, and FIG. 7B shows the result of the metabolite.

From the graph of FIG. 7, the concentrations of kaempferol and metabolites (50% inhibitory concentration: IC ₅₀ ) when the DPPH concentration (absorbance at 517 nm) reached 50% were determined. The IC _{50 for} kaempferol was 23 μM and the IC _{50 for} the metabolite was 30 μM. This shows that even if the 3-position hydroxyl group of kaempferol is substituted with an amino group, it has substantially the same antioxidant activity.

Similarly, the 50% inhibitory concentration (IC ₅₀ ) of quercetin and a compound in which the hydroxyl group at the 3-position was substituted with an amino group was determined to be 7.0 μM for quercetin and 12 μM for its amino form.
<AAPH method>
The antioxidative activity of kaempferol and metabolites is demonstrated by the use of 2,2'-azobis- (2-amidinopropane) dihydrochloride (AAPH), a compound that generates free radicals, of 2'-deoxyguanosine (2'-dG) Evaluation was made by measuring the inhibition (%) of oxide formation.

  When oxidized, 2'-dG becomes 8-oxodeoxyguanosine (8-OHdG), which is read as adenosine during gene transcription. It has been reported that this misreading becomes a mutation and is a direct cause of the occurrence of cancer, arteriosclerosis, diabetes, etc. (Mutat. Res., 267, 1992, 277-290; Free Radic. Biol Med., 26, 1999, 202-226). Therefore, this method of measuring the production inhibition rate of 2′-dG oxide for a test substance is an efficient method for examining the preventive or therapeutic effect of the above-mentioned diseases by the substance.

  Add 5 μl of kaempferol and metabolite to 500 μl of 1.0 mM 2′-dG aqueous solution and 50 mM AAPH, and incubate at 37 ° C. for 1 hour. Then, 8-OHdG, the oxidation product of 2′-dG, is obtained by HPLC. Quantified. The smaller the amount produced, the stronger the antioxidant activity of the test substance.

  The concentration of kaempferol and metabolites in DMSO solution was changed to 0, 0.2, 0.5, 1 and 1.6 mM. A graph plotting the amount of 8-OHdG produced against the concentration of kaempferol or metabolite is shown in FIG. FIG. 8A shows the results of kaempferol, and FIG. 8B shows the results of the metabolites.

From FIG. 8, the concentrations of kaempferol and metabolites (50% inhibitory concentration: IC ₅₀ ) when the production of 8-OHdG was suppressed by 50% were determined. The IC _{50 for} kaempferol was 3.5 μM and the IC _{50 for} the metabolite was 2.5 μM. This also shows that even if the 3-position hydroxyl group of kaempferol is substituted with an amino group, it has equivalent antioxidant activity.

  In the DPPH method, the metabolite has a slightly lower antioxidant activity than kaempferol, whereas in the AAPH method, the metabolite has a slightly higher antioxidant activity than kaempferol. It can be seen that this metabolite has higher antioxidant activity in an aqueous environment such as the human body. From this, it is considered that the metabolite is more likely to express the antioxidant activity in the cell than kaempferol.

Similarly, the 50% inhibitory concentration (IC ₅₀ ) of quercetin and a compound in which the hydroxyl group at the 3-position was substituted with an amino group was determined to be 5.1 μM for quercetin and 11 μM for its amino form.

Examination of the amount of uptake into cells Human hepatoma cell HepG2 is cultured until confluent at 37 ° C, and kaempferol, the 3-position amino body of its chroman skeleton, quercetin and its chroman are obtained in the cell culture to a final concentration of 10 µM. The 3rd-position amino body of the skeleton was added and incubated at the same temperature for 180 minutes.

  The cells were sampled from the culture solution over time, and the nuclei were separated by a homogenization and an established method. That is, cells were collected from the wells of the incubator over time using PBS, and the cells were separated by centrifugation at 3000 rpm for 10 minutes. The cells were homogenized in 0.25 M sucrose, and the nuclei were separated according to a conventional method. . The collected nuclear fraction was divided into two, one of which was extracted with 90% methanol, and the components in the extract were quantified using HPLC.

  HPLC analysis was performed using L-7100 pump (Hitachi, Tokyo, Japan), UV detector (340-370 nm) (Hitachi L-7420) and electrochemical detector (+450 mV) (Rika S 875, Kyoto, Japan) A reverse phase column Capcell pak C18 UG120 (Shiseido, Tokyo, Japan) connected to and was used. The sample injection volume was 20 l, and methanol: 50 mM sodium phosphate buffer (pH 3.3) = 1: 1 was used as the mobile phase. The column temperature was maintained at 35 ° C. and the flow rate was 1 ml / min.

  The antioxidant mass was calculated by the peak height ratio with each sample of 10 mM. Moreover, the antioxidant mass in the sulfatase-treated sample was compared with the untreated amount, and the difference was calculated as the conjugate amount.

  FIG. 9 shows the change in the amount of quercetin, its methyl, its glucuronide / sulfate conjugate and the total amount of these incorporated into the nucleus. In addition, since methylation and conjugation of quercetin are performed by intracellular enzymes, the fact that they were detected in the nucleus indicates that quercetin was taken up into the cells. FIG. 9 shows that quercetin is incorporated into the nucleus as a derivative such as methyl or glucuronide / sulfate conjugate.

Examination of gene oxidation inhibitory activity AAPH was added to the other one of the nuclear fractions collected as described above from the cells cultured with any of the above four types of compounds to a final concentration of 25 mM, at 37 ° C. Incubated for 3 hours.

  DNA was extracted from the cells according to a conventional method, hydrolyzed with endonuclease, and the hydrolyzate was subjected to HPLC to quantify 8-OHdG produced. As described above, 8-OHdG is an oxidation product of guanosine, which is mistaken for adenosine during DNA transcription and causes mutation.

  In detail, Kamendulis, LM, Jiang, J., Xu, Y. and Klaunig, JE (1999) Induction of oxidative damage in rat glial cells by acrylo-nitrile.Carcinogenesis, 20, DNA was extracted by a method partially modified from the method of 1555-1560. That is, AAPH-treated cells were solubilized with TE buffer (10 mM Tris-hydrochloric acid solution, pH 7.4, 10 mM EDTA) containing 0.5% SDS, 500 g / ml ribonuclease A (Sigma) was added, and 50 ° C. Incubated for 30 minutes, added with 500 g / ml proteinase K (Sigma) and incubated at 50 ° C. for 1 hour. Sodium chloride (0.5 M: final concentration) was added thereto, DNA was extracted with 50% isopropanol, and centrifuged at 10,000 g for 15 minutes. The DNA sample was suspended in 200 l of 1 mM EDTA and 15 l of 0.5 M sodium acetate, then treated with nuclease P1 (Sigma) (10 U, 30 min, 37 ° C.), and further 80 l of 0.4 M Tris-HCl. After adding the solution (pH 7.4), it was treated with alkaline phosphatase (Sigma) (3 U, 1 hour, 37 ° C.). After centrifugation (17000 g, 10 min, 4 ° C.), the supernatant was filtered through a 0.2 m filter before HPLC analysis.

  Connect 20 liters of filtrate to L-7100 pump (Hitachi, Tokyo, Japan), UV detector (254 nm) (Hitachi L-7420) and electrochemical detector (+600 mV) (Rika S 875) The analysis was performed using a reverse phase column Capcell pak C18 UG120 (Shiseido). The mobile phase was a mixture of 20 mM potassium phosphate buffer containing 6.5% methanol and 93.5% 0.1 mM EDTA, and the flow rate was 1 ml / min.

The amounts of oxidized products 8-OHdG and 2′-dG were determined by comparison with the peak height of each sample, and the results were expressed as 8-OHdG / 10 ⁵ × 2′-dG.

  FIG. 10 shows the transition of 8-OHdG production when cells are exposed to quercetin. The graph in FIG. 10 shows the 8-OHdG production rate after 0, 30, 60, and 180 minutes after exposure of the cells to quercetin (ratio of 8-OHdG amount at each time point to 8-OHdG amount at 0 minute point). Is shown.

  9 and 10, it can be seen that the production of 8-OHdG is inhibited when the amount of quercetin taken up into the cell is large. This indicates that quercetin suppresses DNA oxidation by AAPH in actual cells.

Examination of the inhibitory effect of Trp-P-2 on mutagenicity When food is cooked, various heterocyclic amines with carcinogenic properties are produced. Japanese people take this in a total amount of about 0.4 to 16 μg / day. ing. Trp-P-2 (3-amino-1-methyl-5H-pyrido [4,3-b] indole) is one of the cyclic amines generated by the decomposition of tryptophan by heating food. Heterocyclic amines are activated by the enzyme CYP1A and then added to genes to induce mutations. Since all heterocyclic amines induce carcinogenesis by the same mechanism, substances that suppress the mutagenicity of Trp-P-2, which is a representative example, show the mutagenicity and carcinogenicity of other cyclic amines. Is also considered to be suppressed. Flavonol is known to suppress DNA mutation caused by heterocyclic amines by regulating CYP1A enzyme activity (Non-patent Document 4).

  Similarly, whether or not the compound of the present invention suppresses the DNA mutation caused by the heterocyclic amine was examined as follows. A mutagenicity test was performed according to a conventional method described in Kanazawa et al., Bioscience, Biotechnology and Biochemistry, 62, 970-977, 1998. That is, the histidine-requiring mutant strain TA98 of Salmonella typhimurium was given a final concentration of 0.1 nmol of Trp-P-2 and S9 mix (rats were given methylcholanthrene and phenobarbital to induce drug metabolism. Into the microsome prepared from the liver, 0.11 nmol of cytochrome P450 1A enzyme was added), kaempferol and its 3-position amino form were added to this bacterial solution at various concentrations, and incubated for 20 minutes. A part of the bacterial solution after the incubation was ingested into a nutrient broth medium and cultured for 2 days, and then sprayed on a synthetic plate medium containing histidine and a synthetic plate medium not containing histidine. Each plate was cultured, and the number of colonies of the revertant strain was counted.

The number obtained by subtracting the number of colonies when nothing is added from the number of colonies when only Trp-P-2 is added is 100, and what is calculated from the values when Trp-P-2 and the test substance are added The number obtained by subtracting the number of colonies without addition of X was defined as X, and the value of (100−X / 100) was defined as antimutagenicity (%). A graph in which antimutagenicity (%) is plotted against the logarithm of the concentration of the test substance is shown in FIG. The concentration showing 50% of the anti-mutagenic (%) was the IC _50. As can be seen from FIG. 11, kaempferol has an IC ₅₀ of 1.3 μM, and its 3-position amino compound has an IC ₅₀ of 1.9 μM, both at low concentrations, indicating a strong mutagenicity-inhibiting effect.

  In addition, the antimutagenicity (%) thus determined is used as an index indicating the antimutagenic agent worldwide. In addition, since Trp-P-2 is a substance that is activated by cytochrome P4501A and exhibits carcinogenicity, it can be seen that kaempferol and its 3-position amino compound have a carcinogenic inhibitory effect.

Further, kaempferol than flavonols (Garanjin, quercetin, morin, fisetin, myricetin) and were examined mutagenic inhibitory effect in the same manner for the 3-position amino compound, IC ₅₀ of the flavonols in the range of 0.6~10μM The IC ₅₀ of the 3-position amino compound was in the range of 0.9 to 12 μM.

Dioxin Toxic Inhibition Expression Dioxin is a compound that is easily taken up into cells and binds to aryl hydrocarbon receptor (AhR) that is inactivated by binding of a protein called heat shock protein 90 in the cell. This binding results in the release of heat shock protein 90 from AhR, which activates (transforms) and translocates AhR into the nucleus. After binding to the nuclear protein called ARNT (Ah receptornuclear translocator), it moves onto the gene and binds to the gene DRE (dioxin response site, also called XRE), thereby expressing the downstream genes. Prompt. As a result, over twenty genes are expressed, but this disrupted expression induces immunodeficiency, teratogenicity and carcinogenesis. A representative expression gene is a gene of CYP1A enzyme described later. Flavonols reversibly inhibit the activation of AhR, the first stage of this toxic expression mechanism (Patent Document 1). Thus, flavonol is the most prominent dioxin toxicity mitigator currently known.

  Whether or not the amino form of kaempferol, which is one of the compounds of the present invention, has a dioxin toxicity suppression effect as in the case of conventionally known flavonols was examined as follows.

  According to the conventional method described in H. Ashida et al., FEBS Letters, 476, 213-217, 2000, TCDD (2,3,7,8-tetrachlorodibenzoate) was obtained in the rat hepatocyte cytoplasm to a final concentration of 1 nmol. After adding dioxane), the aryl hydrocarbon receptor (AhR) was taken out, and the ratio of AhR activated by the binding of TCDD was measured by the gel shift method. At this time, kaempferol and its 3-position amino compound were added to the cytoplasm at various concentrations together with TCDD.

FIG. 12 shows a graph in which the activation rate of AhR is plotted against the logarithm of the concentration of kaempferol and its 3-position amino compound. As can be seen from FIG. 12, kaempferol and its 3-position amino form inhibit AhR activation in a concentration-dependent manner. The concentration that suppresses the activation of AhR by TCDD binding by 50% (IC ₅₀ ) is 1.3 M for kaempferol and 7.9 M for the amino form (12.2 M when AhR is activated by the swerizer method). Thus, it can be seen that the amino form of kaempferol is slightly inferior to kaempferol but suppresses toxicity due to dioxins at a low concentration of about the same level.

Further, when the flavonols other than kaempferol (galandin, quercetin, morin, fisetin, myricetin) and their 3-position amino compounds were similarly examined for their inhibitory effect on the expression of dioxin toxicity, the IC ₅₀ of flavonol was 0.22 to 7.6 μM. The IC ₅₀ of the 3-position amino compound was in the range of 0.55 to 12.2 μM.

Examination of flavonol bioavailability According to a conventional method, human small intestine cancer cells Caco-2 were cultured until they became confluent, and seeded in transwells to prepare monolayer cultured cells. The surface on the upper side of the cell transwell is the surface of the cell on the upper intestinal cavity (villi side) and is called apical. Kaempferol or its 3-position amino compound was added at 2 nmol / well to the apical side, the apical solution in the well was sampled over time, and the concentration of kaempferol or its 3-position amino compound was quantified by HPLC. The HPLC conditions used were those used in Example 12.

  The results are shown as a line graph in FIG. Kaempferol and its 3-position amino form disappeared rapidly and are expected to be taken up into cells. The 3-position amino compound has a higher disappearance rate from the culture supernatant than kaempferol.

  Further, the concentration of kaempferol and its 3-position amino glucuronide-sulfate conjugate is shown as a bar graph in FIG. 13 (A). These conjugates increase with time, and it can be seen that kaempferol and its 3-position amino form are excreted in the form of conjugates on the small intestinal cavity side. In addition, the amount of conjugate produced is less at the 3-position amino compound than kaempferol.

  From these results, it is understood that the 3-position amino compound is taken into the cell more rapidly than kaempferol, and is conjugated and excreted as feces or urine.

  The cells in the wells were sampled over time and homogenized to extract the cell contents, and the amount of kaempferol or its 3-position amino body per well was quantified by HPLC. The HPLC conditions used were those used in Example 12. The results are shown as a line graph in FIG. The rate of uptake into cells was approximately the same for amino and kaempferol at the beginning of incubation, but the intracellular concentration of amino was higher with time.

  In addition, the amount of kaempferol or its 3-position amino glucuronide-sulfate conjugate per well is shown by a bar graph. The amino form has a lower conjugate production rate than kaempferol. From these facts, it can be seen that the amino form is more easily taken up into the cell than kaempferol and hardly becomes a conjugate, and as a result, the effect on AhR and CYP1A in the cell is effectively maintained.

  The blood side (vasola terrayl side) solution of the cells in the transwell was sampled over time, and the amount of kaempferol or its 3-position amino body per well was quantified using HPLC. The HPLC conditions used were those used in Example 2. The results are shown as a line graph in FIG. From FIG. 13 (C), it can be seen that the amino form is excreted to the batholaterrayl side at a higher rate than kaempferol.

  Moreover, the density | concentration of these conjugates in a bathola terrayl side solution is shown with a bar graph. It can be seen that the amino conjugate is transferred to the basolateil side at an extremely high speed compared to the kaempferol conjugate.

  In this way, the amino form migrated to the basolateil side in a larger amount in both the free form and the conjugate form compared to kaempferol, and the amino form migrated from the small intestinal cells into the blood on the serosa side in vivo. It turns out that it is easy.

  From FIGS. 13 (A), (B), and (C), amino is more easily taken into the small intestine than kaempferol, has a long residence time in the small intestine, and has a high blood concentration, that is, high bioavailability. I understand.

  Thus, the 3-position amino form of the flavonol of the present invention is slightly inferior to the corresponding flavonol, but has substantially the same DNA oxidative damage-inhibiting action, DNA mutation-inhibiting action, dioxin toxicity expression-inhibiting action, Since bioavailability is higher than that of the corresponding flavonol, it is considered that when a human ingests it as a medicine or food, it exhibits a physiological activity equivalent to or higher than that of the corresponding flavonol.

  The compound represented by the general formula (1) of the present invention includes an anticancer composition, an immunodeficiency prevention composition, a teratogenicity prevention composition, an anti-inflammatory composition, and a composition for preventing or treating arteriosclerosis. Can be suitably used as an active ingredient of a composition for preventing diseases and diabetes.

It is a figure which shows the UV spectrum of a metabolite and kaempferol. It is a figure which shows the FT-IR spectrum of a metabolite and kaempferol. (A) shows a ¹ H-NMR spectrum of kaempferol, (B) is a diagram showing ¹ H-NMR spectrum of the metabolite. (A) shows a ¹³ C-NMR spectrum of kaempferol, (B) is a chart showing ¹³ C-NMR spectrum of the metabolite. It is a figure which shows the FAB-MS spectrum of kaempferol. It is a figure which shows the FAB-MS spectrum of the metabolite of kaempferol. It is the graph which plotted DPPH concentration (%) with respect to kaempferol concentration (A) and its metabolite concentration (B). It is the graph which plotted the production amount (%) of 8-OHdG with respect to kaempferol concentration (A) and its metabolite concentration (B). It is a graph which shows the uptake | capture amount in a cell with time, such as a quercetin and its conjugate. It is the graph which plotted the production amount (%) of 8-OHdG with respect to the time for quercetin to act on DNA. It is the graph which plotted the number of reversion | reversion mutants of Salmonella typhimurium, ie, the number by which the variation | mutation was suppressed with respect to the logarithm of the density | concentration of kaempferol and its 3rd-position amino body. It is the graph which plotted the activation rate of AhR with respect to the logarithm of the density | concentration of kaempferol and its 3rd-position amino body. (A) line graph is a graph showing the transition of the amount of kaempferol and its 3-position amino body in the liquid on the small intestinal cell upper space side, the bar graph is a graph showing the transition of the amount of these conjugates, The line graph in (B) is a graph showing the transition of the amount of kaempferol and its 3-position amino body in small intestinal cells, the bar graph is the graph showing the transition of the amount of these conjugates, and the line in (C) A graph is a graph which shows transition of the quantity of kaempferol and its 3-position amino body in the fluid of the serosa side of small intestinal cells, and a bar graph is a graph which shows transition of the quantity of these conjugates.

Claims (21)

The compound represented by the following general formula (1).
The following general formula (2)
(In the formula, R ¹ represents a hydroxyl group or a linear or branched alkoxy group having 1 to 5 carbon atoms, and R ² represents a hydrogen atom, a hydroxyl group or a linear or branched group having 1 to 5 carbon atoms. Represents an alkoxy group.)
A compound represented by the following general formula (3):
(In the formula, R ¹ represents a hydroxyl group or a linear or branched alkoxy group having 1 to 5 carbon atoms, and R ² represents a hydrogen atom, a hydroxyl group or a linear or branched group having 1 to 5 carbon atoms. Represents an alkoxy group.)
A step of incubating the compound of the present invention with a mammalian small intestine surface cell homogenate; and a step of recovering the compound of the general formula (2) . The production method according to claim 2 , wherein, in the general formulas (2) and (3) , R ¹ represents a hydroxyl group and R ² represents a hydrogen atom. The following general formula (1)
A compound represented by the following general formula (4):
And a step of allowing the compound of the above formula (1) to recover; and a step of recovering the compound of the general formula (1). The method according to claim 4 , wherein the step of leaving the compound of the general formula (4) in the presence of ammonia is carried out under pressure. A gene oxidation inhibitor comprising the compound according to claim 1 . The dioxin toxicity expression inhibitor which consists of a compound of Claim 1 . An anticancer agent comprising the compound according to claim 1 . An immunodeficiency preventive agent comprising the compound according to claim 1 . A teratogenicity preventive agent comprising the compound according to claim 1 . An anti-inflammatory agent comprising the compound according to claim 1 . A preventive or therapeutic agent for arteriosclerosis comprising the compound according to claim 1 . A preventive or therapeutic agent for diabetes comprising the compound according to claim 1 . A composition for inhibiting gene oxidation, comprising the compound according to claim 1 as an active ingredient. The composition for dioxin toxicity expression suppression which uses the compound of Claim 1 as an active ingredient. An anticancer composition comprising the compound according to claim 1 as an active ingredient. A composition for preventing immunodeficiency comprising the compound according to claim 1 as an active ingredient. A composition for preventing teratogenicity comprising the compound according to claim 1 as an active ingredient. An anti-inflammatory composition comprising the compound according to claim 1 as an active ingredient. A composition for preventing or treating arteriosclerosis comprising the compound according to claim 1 as an active ingredient. A composition for preventing or treating diabetes comprising the compound according to claim 1 as an active ingredient.